RESUMO
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (DeltaPMM and DeltaDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana DeltaDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana DeltaPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
Assuntos
Leishmania mexicana/enzimologia , Leishmania mexicana/patogenicidade , Manosiltransferases/genética , Fosfotransferases (Fosfomutases)/genética , Virulência/genética , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Clonagem Molecular , Dolicol Monofosfato Manose/metabolismo , Regulação para Baixo , Citometria de Fluxo , Deleção de Genes , Marcação de Genes , Glicoconjugados/metabolismo , Glicosilação , Guanosina Difosfato Manose/metabolismo , Leishmania mexicana/genética , Macrófagos/parasitologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fenótipo , Homologia de Sequência de AminoácidosRESUMO
The polar glycoinositol phospholipids (GIPLs) of a Trypanosoma species that belongs to the Schizotrypanum subgenus were purified by reversed-phase and normal-phase liquid chromatography and analysed by negative-ion mode electrospray-mass spectrometry (ES-MS). The phosphatidylinositol moieties were released by nitrous acid deamination and identified as ceramide- and alkylacylglycerol-containing species. The structures of the GIPLs were determined using chemical treatments, sequential exoglycosidase digestions and positive-ion mode ES-MS-MS. All of the GIPLs were based on the same Man alpha1-2Man alpha1-2Man alpha1-6Man alpha1-4(NH2-CH2CH2-HPO3-)GlcN-PI core with single terminal Galf residue substitutions either on the terminal nonreducing Man or on the second alphaMan residue from the inositol and with either ethanolamine phosphate or 2-aminoethylphosphonate on the third alphaMan residue from the inositol. The T. (S.) dionisii GIPLs are compared with those of T. (S.) cruzi, a closely related species of the Schizotrypanum subgenus.
Assuntos
Quirópteros/parasitologia , Glicosilfosfatidilinositóis/química , Trypanosoma cruzi/química , Trypanosoma/química , Animais , Sequência de Carboidratos , Cromatografia/métodos , Glicosilfosfatidilinositóis/isolamento & purificação , Metilação , Dados de Sequência Molecular , Análise Espectral/métodosRESUMO
The major diagnostic antigen of Paracoccidioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain. This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence. It contains a neutral high-mannose core (Man7GlcNAc2) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached. A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide. The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit. The complete structure was determined by methylation analysis, 1H-NMR, mass spectrometry, acetolysis and mannosidase degradation. Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex18GlcNAc2 to Hex10GlcNAc2 which accounts for the diffuse migration of the gp43 in polyacrylamide gels. The average size of the most frequent subtype is Hex13.6GlcNAc2. Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.
Assuntos
Antígenos de Fungos/química , Proteínas Fúngicas , Glicoproteínas/química , Oligossacarídeos/química , Paracoccidioides/química , Acetilação , Sequência de Carboidratos , Cromatografia , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Paracoccidioides/imunologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/diagnóstico , Análise de Sequência/métodosRESUMO
The major acceptors of sialic acid on the surface of metacyclic trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to the parasite membrane via glycosylphosphatidylinositol anchors. Here we have compared the lipid and the carbohydrate structure of the glycosylphosphatidylinositol anchors and the O-linked oligosaccharides of the mucins isolated from metacyclic trypomastigotes and noninfective epimastigote forms obtained in culture. The single difference found was in the lipid structure. While the phosphatidylinositol moiety of the epimastigote mucins contains mainly 1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol, the phosphatidylinositol moiety of the metacyclic trypomastigote mucins contains mostly (approximately 70%) inositol phosphoceramides, consisting of a C18:0 sphinganine long chain base and mainly C24:0 and C16:0 fatty acids. The remaining 30% of the metacyclic phosphatidylinositol moieties are the same alkylacylphosphatidylinositol species found in epimastigotes. In contrast, the glycosylphosphatidylinositol glycan cores of both molecules are very similar, mainly Man alpha 1-2Man alpha 1-2Man alpha 1- 6Man alpha 1-4GlcN. The glycans are substituted at the GlcN residue and at the third alpha Man distal to the GlcN residue by ethanolamine phosphate or 2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote mucin-like molecules, released by beta-elimination with concomitant reduction, are identical to the structures reported for the epimastigote mucins (Previato, J. O., Jones, C., Gonçalves, L. P. B., Wait, R., Travassos, L. R., and Mendoça-Previato, L. (1994) Biochem. J. 301, 151-159). In addition, a significant amount of nonsubstituted N-acetylglucosaminitol was released from the mucins of both forms of the parasite. Taken together, these results indicate that when epimastigotes transform into infective metacyclic trypomastigotes, the phosphatidylinositol moiety of the glycosylphosphatidylinositol anchor of the major acceptor of sialic acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.
Assuntos
Glicosilfosfatidilinositóis/química , Lipídeos/química , Mucinas/química , Trypanosoma cruzi/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Diferenciação Celular , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/química , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 Man alpha 1-4 GlcN alpha 1-6myo-inositol, and Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 (GalNAc beta 1-4) Man alpha 1-4 GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
Assuntos
Acetilcolinesterase/biossíntese , Órgão Elétrico/química , Glicosilfosfatidilinositóis/química , Torpedo , Animais , Sequência de Carboidratos , Cromatografia Gasosa-Espectrometria de Massas , Espectroscopia de Ressonância Magnética , Dados de Sequência MolecularRESUMO
The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glcalfa1-2 Manalfa1-2 Manalfa1-6 Manalfa1-4 GlcNalfa1-6myo-inositol, and Glcalfa1-2 Manalfa1-2 Manalfa1-6 (GalNAcß1-4) Manalfa1-4 GlcNalfa1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule